Volume 5 Number 2 February 1978
Nucleic Acids Research
A rapid method for the isolation of circular DNA using an aqueous two-phase partition system R. OhIsson+, C.C. Hentschel* and J.G. Williams*
+National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1 AA and *Imperial Cancer Research Fund, Burtonhole Lane, Mill Hill, London, NW7 1AD, UK Received 23 December 1977 ABSTRACT A method of isolating circular plasmid DNA from cleared lysates of E.coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics of supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dyecentrifugation technique and plasmid DNA of comparable purity and yield is obtained.
INTRODUCTION
Mojecular cloning has become a widely used technique for the isolation of specific nucleic acid sequences and bacterial plasmids are the vector of choice for many cloning experiments. However, methods of preparing plasmid DNA have evolved very little since the development of caesium
chloride-ethidium bromide centrifugation of cleared bacterial lysates (1). While this technique provides a high degree of purification it is expensive in both centrifuge time and materials and there is a clear application for a rapid method of purification. We have developed a technique which yields essentially pure plasmid DNA within minutes of obtaining a phenolized cleared bacterial lysate: The technique depends on the fact that plasmid DNA, being a closed circular molecule, displays mono-molecular re-annealing kinetics (2). After denaturation of a mixture of plasmid and E.coli chromosomal DNA, plasmid DNA renatures very rapidly while chromosomal DNA remains single stranded. A two phase partition system is then used to purify duplex plasmid DNA away from single stranded chromosomal DNA and bacterial RNA. This technique has proved particularly useful in the screening of large numbers of transformed clones to determine the restriction enzyme pattern of their plasmid DNA. Since there is no inherent limitations on the scale of the procedure
C Information Retrieval Limited 1 Falconberg Court London WI V 5FG England
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Nucleic Acids Research it
may
also
prove
useful in the preparation of
very
large quantities of
plasmid DNA. MATERIALS AND METHODS
Poly-ethylene glycol 6000 (PEG)
was
(England) and Dextran 500 (lot No. 3447)
obtained from BDH Chemicals was
Fine Chemicals (Sweden). E. Coli ribosomal RNA, transfer
chromosomal DNA The
restriction
were
RNA and
obtained from Miles Laboratories Ltd.
enzyme
Hae III
was a
Ltd.
purchased from Pharmacia
(England).
gift from Dr. B. Griffin (Imperial
Cancer Research Fund, London, England) and SV40 DNA
was
a
gift from
Dr. R. Kamen (Imperial Cancer Research Fund, London, England). Plasmid
DNA
prepared from late exponential phase cultures treated for 16 hr
was
with chloramphenicol at 170 pg/ml to amplify the plasmid DNA. Cleared lysates
(3).
were
The
prepared by the Triton X-100 lysis procedure of Katz et al.
cleared lysates
for 30' at 37 C.
were
then treated with Proteinase K at 100 ig/ml
Finally, they
were
extracted twice with phenol and twice
with chloroform-isoamyl alcohol (99 to 1) and precipitated with 2 volumes of ethanol. (Chloramphenicol In
a
was
obtained from Sigma and Proteinase K from Merck)
typical experiment to purify plasmid DNA for restriction
analysis, the phenolized cleared lysate of
5 ml culture
a
was
enzyme
resuspended
(after ethanol precipitation) in 0.2 ml of TE-buffer (10 mM Tris pH 8.0, 1 mM EDTA) in
a
Microfuge tube.
roughly 25 lig/ml.
The
sample
was
This gives
a
plasmid DNA concentration of
heated for 2 minutes at 1000C to denature
chromosomal DNA and rapidly cooled in dry ice-ethanol. Then 500 pl of dextran 500 (16.8% W/W), 250 pl of PEG (18.4% W/W) and 20
sodium phosphate buffer pH 6.8,
were
added at 40C.
The
adjusted to 1 ml with water and the whole stirred with
a
il
of 0.5 M
volume was then
vortex mixer. It
is important to note that the exact concentrations of the components of the two-phase system (this was determined by freeze druing
a
weighed aliquot
of the stock solution), the pH of the buffer and the temperature (4 C) are
critical factors in determining the separation achieved (for reviews
of two-phase separation techniques the polymer phase
fuge for
1
was
see references 4 and 5).
accelerated by centrifugation
minute at 4 C.
Then 450
Separation of
in a Beckman Micro-
4l of the upper, PEG rich, phase was
recovered and transferred to a fresh tube. After addition of NaCl to 0.2 M, DNA
was
collected by precipitation with two volumes of ethanol. This
procedure also precipitates the phosphate and a small amount of PEG but this does not interfere with subsequent restriction enzyme analysis. (An alternative procedure which avoids co-precipitation of phosphate and PEG
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Nucleic Acids Research is cetyl trimethyl asnonium bromide (CTAB) precipitation (8)). Pellets were
washed twice in 100% ethanol, dried and taken
Restriction analysis
was
performed using the
subsequent electrophoresis scopy
was
performed
on
on
up
in
a
small volume of
enzyme Hae III
3.5% acrylamide gels (6).
DNA spread by the
aqueous
H20.
with
Electron micro-
procedure of Davies
et al. (7).
RESULTS
The principal components in
a
phenol extracted, cleared lysate of
ribosomal RNA and transfer RNA; plasmid DNA, when
E. coli
are
forms
relatively small fraction of the total nucleic acid.
a
plasmid DNA there is normally DNA. This depending
on
a
present,
In addition to
roughly equivalent amount of chromosomal
the efficiency of the chloramphenicol amplification
step and the effectiveness of the final centrifugation step in the lysis
procedure.
In order to determine the potential purification of plasmid
DNA achievable in
a
two phase partition system,
a
model experiment
was
performed using purified E. coli RNA and DNA. The two phase system was optimized as described by Alberts (5) and the results are presented in Table la. As would be expected from previous results (5,6) only double stranded nucleic acids
were
found to
any
significant extent in the
upper
PEG rich phase. Thus partition under these conditions should produce
substantial purification of plasmid DNA
away
a
from the principal
contaminant E. coli ribosomal RNA. We reasoned that additional purification would result from heat
denaturation of the nucleic acids prior to the partition, since the closed circular plasmid DNA will re-anneal rapidly under these conditions (2),
and hence remain in the PEG phase. (A similar approach has been used to purify other rapidly re-annealing species (9,10). This proved to be correct (Table la) and, after heat treatment, chromosomal DNA was almost totally removed from the PEG phase. A significant amount of transfer RNA remains in the PEG phase, irrespective of heat treatment, presumably because of the large amount of intramolecular base pairing which causes rapid reannealing. For most purposes the presence of transfer RNA presents
no
problem and we decided to attempt plasmid purification using this procedure. an
Table lb shows the degree of purification that can be achieved when artifical mixture of plasmid DNA, chromosomal DNA and ribosomal RNA are
subjected to phase separation. Using one cycle of purification a
17-fold
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Nucleic Acids Research enrichment was obtained, and after a second partition of the PEG phase with dextran this was increased to 35 fold. The yield of plasmid
DNA was
79% after one cycle of purification and 61% after two cycles. For restriction analysis the degree of purity and yield of plasmid DNA after a single phase extraction was more than adequate, and all subsequent
preparations were purified in this way.
The use of this technique to purify plasmid DNA from bacterial lysates is illustrated in Fig. 1.
A series of bacterial clones, each
containing PMB9 plasmid with a different eukaryotic DNA sequence inserted at the ECoRI site, were grown up and chloramphenicol amplified. Phenol TABLE la
Distribution of various E.coli nucleic acids after two-phase partition % of input in upper phase (PEG)
Sample Native
After heating at 1060C for 2 minutes
1.8
Ribosomal RNA
Transfer RNA
37
Chromosomal DNA
61
3H
79
Plasmid (PMB9) DNA
1.6 37
2.5 78
A 1 ml phase partition system was used (see Materials and Methods) and the input of each, non-isotopically labelled, nucleic acid was about six 0D260 units. The 3H labelled PMB9 DNA was prepared by standard dyecentrifugation procedure and had a specific activity of 200 cpm per jig; 1,000 cpm was used in each partition. TABLE lb Distribution of an artifical mixture of nucleic acids after two-phase partition - fold purificatio
% of total OD units recoverea in upper polymer phase
% recovery of 3H PMB9
single two-phase partition
4.7
79
16.7
The same samples after a second two-phase partition
1.7
61
35.1
Samples subjected to a
The artificial mixture contained, by weight 50 parts chromosomal DNA, 50 parts ribosomal RNA and 1.4 parts 3H PMB9 (comprising 5 jig). A 1 ml phase partition system was used and the second partition was performed by adding 450 pl of dextran and 20 jil of phosphate buffer to the PEG phase. The theoretical maximum purification is 72.3 fold.
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Nucleic Acids Research
a
b
c
d
e
f
g
hi
Figure 1. The total nucleic acid from phenolized, cleared lysates of eight bacterial cultures (20 ml), each containing a fragment of eukaryotic DNA of undetermined length, was resuspended in 1 ml of distilled H20. Two hundred microlitre aliquots of each preparation were subjected to a single two phase partition and ethanol precipitated. Each pellet was then dissolved in 20 4l of H20, restricted with Hae III and run on 3.5% acrylamide gel (slots b to h). One hundred microlitre aliquots of the preparations run in slots b and h were ethanol precipitated and restricted without purification (slots a and i). The outside slots were loaded with an SV40 marker DNA digested to completion with Hin III.
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Nucleic Acids Research
Figure 2. A phenolized cleared lysate of E.coli containing the plasmid PMB9 was subjected to a single two phase partition. Samples removed before and after purification were prepared for electron microscopy under the aqueous conditions of Davies et al. (8). The sample removed before purification showed a large amount of granular material, assumed to be RNA, which completely obscured DNA molecules (results not shown). The field shown is quite typical of the purified preparation. extracted cleared lysates were then prepared and an aliquot of each preparation was subjected to aqueous two-phase partition, restricted with Hae III and analyzed by electrophoresis on 3.5% acrylamide gels. For two of the preparations, an aliquot of unpurified nucleic acid was
similarly analyzed (Fig. 1). The majority of the nucleic acid in the unpurified samples is RNA, which totally obscures the smaller Hae III restriction fragments of the plasmid DNA. There is also a significant amount of chromosomal DNA, giving rise to a restriction enzyme pattern which partially obscures the large restriction enzyme fragments of the plasmid DNA. After two-phase partition plasmid DNA appears to be pure except for a significant amount of tRNA which runs near the end of the gel. Gel electrophoresis, however, is not sensitive enough to detect a low level contaminant of heterogeneous size (such as restriction fragments of chromosomal DNA). Therefore, similar samples were analyzed by electron microscopy. Before phase partition the presence of large amounts of RNA made it impossible to quantitate the various species. After phase 588
Nucleic Acids Research partition (Fig. 2), in a total of 95 molecules examined, there were 92 supercoils, and 3 linear molecules of lengths different from that of the
plasmid DNA, this indicates a very high degree of purification with respect to chromosomal DNA. DISCUSSION
The great virtue of the aqueous two-phase separation technique presented here is the speed with which a quite highly purified preparation
of plasmid DNA can be prepared with a minimum expense in materials and equipment. Once a phenolized cleared lysate has been obtained, the
entire procedure takes less than one hour.
This compares with two or
three days of centrifugation to obtain caesium chloride purified plasmid, which subsequently has to be purified to remove ethidium bromide and other components of the lysis and centrifugation solutions. The small scale on which the procedure can be performed means that plasmid DNA can be prepared from a few ml. of E.coli and this reduces the potential hazards associated with growing large amounts of an uncharacterized eukaryote cloned DNA. Another small scale plasmid enrichment procedure has been reported (11)
but this requires an RNAase step and contaminating chromosomal DNA is not removed.
Our technique can easily be scaled up to prepare very large
quantities of plasmid DNA in which case the alkali denaturation procedure of Sunmmers and Szybalski (9) is convenient since no boiling step is
required. The procedure is of course very suitable for dealing with large numbers
of samples. We have found this particularly useful when screening individual "cDNA" clones to determine the size of the eukaryotic DNA insert. This
being a common requirement of the cloning procedure, even if size selected cDNA is used for the cloning. (Fig. 1 illustrates such a screen). However, the technique clearly has application in all situations where contamination by E.coli tRNA and a small amount of E.coli chromosomal DNA
constitutes no problem.
The data in Table lb is particularly impressive
in view of the very high ratio of chromosomal DNA to plasmid DNA in these This is much higher than that actually found in
samples (49 to 1.4).
chloramphenicol amplified cleared lysates (which we estimate as being about 1 to 1). This indicates that the method can probably be used to purify plasmids, which cannot be chloramphenicol amplified (especially if two cycles of phase separation are used). Also of course it is applicable to any closed circular or spontaneously
re-annealing DNA and
we have
recently used this technique to purify mitochondrial DNA (C.J. Coote and
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Nucleic Acids Research R. Ohlsson, unpublished results).
ACKNOWLEDGEMENTS We
are
grateful for the excellent technical assistance of Mrs Rita
Harris and Mrs M. Lloyd. Mrs Rita Tilly.
Electron microscopy analysis
We also wish to express
providing valuable advice and-laboratory
our
was
performed by
gratitude to Dr. J. Tata for
space.
Valuable advice has also
been given by Professor P.A. Albertsson. Dr. R. Ohlsson is
an
EMBO fellow.
REFERENCES
2
Radloff, R., Bauer, W. and Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57: 1514-1522 Helinski, D.R. and Clewell, D.B. (1971) Ann. Rev. Biochem. 40:
3
Katz, L., Kingsbury, D.K. and Helinski, D.R. (1973) J. Bacteriol.
4
Albertsson, P.A. (1971) In: Partition of cell particles and macromolecules. Wiley-Interscience, New York, 151-159 Alberts, B. (1967) In: Methods in Enzymology. Vol 12A (ed. S.P. Colowick and N.O. Kaplan) Academic Press, New York, 566 Maniatis, T., Jeffrey, A. and van de Sande, H. (1975) Biochemistry
1
899-942 114:577 5 6
14:3787-3794 7
8
9 10 11
Davies, R.W., Simon, M. and Davidson, N. (1971) In: Methods in Enzymology, vol 21, Part D 413-428 Stehelin, D., Guntaka, R.V., Varmus, H.E. and Bishop, J.M. (1976) J. Mol. Biol. 101:349 Summers, W.C. and Szybalski, W. (1967) J. Mol. Biol. 26:107-122 Patterson, J.B. and Stafford, D.W. (1971) Biochemistry 9:1278 Meagher, R.D., Tait, R.D., Betlack, M. and Boyer, H.W. (1977) Cell
10:521-536
590